SUMMARY ? PROJECT 3 (AIFANTIS) Recent studies have offered the first comprehensive maps of three-dimensional (3D) chromosomal interactions. The 3D structure of chromatin is defined in part by the organization of chromatin into highly conserved topologically associating domains (TADs). Studies presented in Project 1 and 2 directly address the role of key ?structural? elements of TAD boundaries (CTCF/Cohesin) in human cancer, including B cell and T cell malignancy (leukemia and lymphoma). In Project 3, we will test the hypothesis that 3D chromatin structure is not only affected by CTCF/Cohesin alterations but also by mutations that affect specific epigenetic regulators and by oncogenic transcription factors. For these studies, we will use T cell leukemia (T-ALL), as a model of study. We will test whether T-ALL oncogenes (the transcription factor NOTCH1, the main driver in this disease, mutated or activated in 90% of human T-ALL) use 3D DNA looping events to induce expression of gene- targets and non-protein coding RNAs that control the function of leukemia cells, including cells that can initiate the disease (leukemia initiating cells), that are characterized by the overexpression of the NOTCH1 transcriptional target MYC. In addition to oncogenic (NOTCH1, MYC) activation, chromosomal topology can also be influenced by epigenetic regulators, and it was shown that T-ALL is a disease characterized by recurrent inactivating mutations in genes that can affect DNA and histone modifications, including genes that affect DNA methylation (DNMT3A), promoter (EZH2) and enhancer (EP300) activity. We thus hypothesize that in human leukemia oncogenes (NOTCH1) and tumor suppressors (DNMT3A, EZH2, EP300) cause aberrant 3D chromatin organization changes and that targeted drug treatments are able to correct these defects. We test this hypothesis in three Aims. Aim 1 assesses the ability of oncogenes like NOTCH1 to directly alter CTCF/Cohesin distribution leading to aberrant chromosomal architecture. Aim 2 tests the hypothesis that drugs that target either oncogenic signaling pathways (NOTCH pathway inhibitors) or altered epigenetic states (BET inhibitors, targeting active H3K27ac-marked areas) can correct 3D chromosomal structure. Finally, Aim 3 focuses on potential effects of selected T-ALL somatic mutations targeting epigenetic regulators and examines their impact on chromosomal topology. We believe that these studies will complement Projects 1 and 2 that focus on the impact of CTCF/Cohesin alterations in blood cancer and generate new paradigms of gene expression regulation in human leukemia.